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Transgenic product low-density gene chip detecting method

A gene chip and detection method technology, applied in the field of detection of genetically modified products using low-density gene chips, can solve the problems of human and environmental hazards, lack of specificity and sensitivity, and achieve the effect of reducing the mismatch rate

Inactive Publication Date: 2005-02-16
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of polygenic PCR products still uses the traditional gel electrophoresis analysis method, which is simple and easy to implement, but lacks specificity and sensitivity, and is likely to cause harm to humans and the environment

Method used

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  • Transgenic product low-density gene chip detecting method
  • Transgenic product low-density gene chip detecting method
  • Transgenic product low-density gene chip detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Selection of appropriate primers and probes is the key to successful DNA analysis and detection. Aiming at the sequences of the 9 most commonly used exogenous DNAs and control genes (such as 18S ribosomal RNA, soybean lectin endogenous gene and maize invertase endogenous gene) in transgenic products, various parameters are adjusted through repeated combinations, and the software Primer Express TM Search and analyze with OLIGO 4.0, and compare each oligonucleotide, and select primers and probes that meet the requirements. The selected primers and related information are shown in Table 1, and the probes used and related information are shown in Table 2.

[0039] Primer number

Primer sequence (5'-3')

target DNA

18S-1

TAA TAG AGC AAT GAA CAG TCG G

18S-2

CTT TCA ACA ACG GAT CTCTT G G

18S ribosomal RNA

lec-1

GTG CTA CTG ACC AGC AAG GCA AAC TCA GCG

lec-2

GAG GGT TTT GGG GTG CCG TTT TCG TCA AC

Soybean lec...

Embodiment 2

[0048] Using PixSys TM Model 5500 automatic spotting instrument, designed as a 16×4 array, contains 14 detection target probes, 2 negative controls, and 4 spots for each probe (see figure 1 ). A human gene that has almost no homology with the detection probe is set on the chip as a negative control to exclude the interference of non-specific hybridization during the hybridization process.

Embodiment 3

[0050] Chip hybridization refers to the process in which fluorescently labeled samples react with probes on the chip and generate a series of information. Factors that affect the formation of chip hybridization double strands include target concentration, hybridization solution components, and hybridization temperature. Selecting appropriate conditions can make most of the hybridization reactions in the best condition, that is to say, make as many correct pairs as possible. Missed, crosses with mismatches are minimized. The listed hybridization procedures (including reaction conditions such as probe modification, target concentration, hybridization temperature, hybridization time, and slide washing) have been optimized, such as amination of the 5' end of the probe and ligation of 10 thymines After the connecting arm composed of nucleosides (T), the immobilization efficiency of the probe on the slide can be significantly improved, and the hybridization effect can be improved; t...

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Abstract

The low-density gene chip detection method of transgenic product relates to a detection method for transgenic product by uisng low-density gene chip. Said method includes the following steps: selecting primer and probe; processng chip carrier, preparing chip; dissolving probein ultrapure water; adding sample application liquid; fixing the probeon the treated chip carrier, sampling; multigene PCR amplification; labeling MPCR product; chip hybridization experiment of MPCR product; scanning analysis and data processing. As compared with traditional detection method said invention has the characteristics of quick seep, high efficiency and high automation level, and can simultaneously make detection and analysis of common 9 exogenous DNA sequences of promoter, terminator, screening marker gene, weedicide-resisting gene and insect-resisting gene, etc. in transgenic products.

Description

technical field [0001] The invention relates to a detection method for genetically modified products, in particular to a method for detecting genetically modified products using a low-density gene chip. Background technique [0002] With the rapid development of genetically modified crops, the environmental safety and food safety of genetically modified products have attracted widespread attention from governments and the public (Anklam E, Gadani F, Heinze P, et al, Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur. Food Res. Technol., 2002, 214, 3-26) In order to protect consumers' right to know and right to choose, to ensure the safety of people's dining tables, and to ensure the healthy development of international trade in agricultural products, countries around the world Both have increased research investment in the detection and safety evaluation of genetically modified product...

Claims

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Application Information

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IPC IPC(8): G01N33/48G01N35/00
Inventor 苏文金刘光明宋思杨
Owner XIAMEN UNIV
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